Immunotherapeutic agent

ABSTRACT

Compounds for use in the treatment of sepsis and/or the prevention or treatment of post-sepsis syndrome.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 62/440,353 filed Dec. 29, 2016, the disclosure ofwhich is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention provides compounds for use in the treatment ofsepsis and/or the prevention or treatment of post-sepsis syndrome.

BACKGROUND OF THE INVENTION

Sepsis, a potentially life-threatening condition triggered by aninfection or injury, is the result of an overwhelming inflammatory hostresponse to bacterial infection. During sepsis, the body's immune systemis dysregulated as it tries to fight an infection, and this can reduceblood supply to vital organs such as the brain, heart and kidneys whichcan cause multiple organ failure and ultimately death.

It had long been believed that sepsis merely represented an exaggerated,hyperinflammatory response that led to inflammation-induced organinjury, but recent data indicate that substantial heterogeneity existsin septic patients' inflammatory response, with some appearingimmuno-stimulated, whereas others appear suppressed. It is clear thatmultiple pathological mechanisms contribute to sepsis.

The current view of sepsis includes the concept of a SystemicInflammatory Response Syndrome (SIRS) that has been induced by aninfection. Recent data suggests that an appropriate inflammatoryresponse allows elimination of invading microorganisms without causingdamage to tissues, organs, or other systems. However, dysregulatedinflammation prevents this elimination and allows sepsis to occur bypermitting the physiological alterations that manifest as the SIRS.Macrophage activation is thought to play a central role in theinitiation and propagation of the systemic inflammatory response.

Inflammatory cells, and monocytes and macrophages in particular, play acentral role in the response to invading pathogens and in thepathogenesis of sepsis (Van Amersfoort, E. S. et al. (2003) Clin.Microbiol. Rev. 16(3):379-414). Macrophages in particular have beendirectly implicated in numerous pathogenic mechanisms that cause organdamage in the septic patient, including for example cardiac infiltrationleading to heart disease (Cuenca, J. et al. (2006) Am. J. Pathol.169(5):1567-1576)

Despite intense efforts, sepsis remains a serious clinical problemdespite improvements in critical care management. Despite widespreadavailability of antibacterial therapies and the development of improvedimmunotherapies, there remains a need to improve the efficacy oftreatment of sepsis.

SUMMARY

The present invention provides compositions for the treatment of sepsis.More specifically the invention provides synthetic fatty acids andmono-, di- and tri-esters of the fatty acid and 1,2,3-propanetriol,which are useful in treating sepsis.

The present invention is based on the surprising finding by theinventors that previously described esters of 1,2,3-propanetriol withone or more C₁₁ to C₂₄ fatty acids, having at least one fatty acidhaving only one double bond at the C10-C11 position preventedlipopolysaccharide-stimulated secretion of the proinflammatory cytokineIL-6 from freshly isolated peritoneal macrophages. Such compoundsdown-regulate expression of proinflammatory cytokine and chemokineligand mRNAs in activated macrophages and inhibit the pro-inflammatorysignaling of activated macrophages in vitro. Compounds of the inventionare therefore useful in inhibiting the proinflammatory cascade thatcontributes to the pathology of sepsis.

In a first aspect, the invention provides an ester of 1,2,3-propanetriolwith one or more fatty acids of at least C11, wherein at least one fattyacid has a double bond at the C10-C11 position, for use in the treatmentof sepsis.

The compounds of the first aspect can be used to inhibit proinflammatorysignaling by activated macrophages in sepsis, and this can alloweffective treatment of infection underlying the sepsis while preventingthe organ damage that it produces.

In a second aspect, the invention provides a fatty acid of at least C11comprising a double bond between C10 and C11 for use in the treatment ofsepsis or and/or the prevention or treatment of post-sepsis syndrome.

The compounds of the second aspect can be used to inhibitproinflammatory signaling by activated macrophages in sepsis, and thiscan allow effective treatment of infection underlying the sepsis whilepreventing the organ damage that it produces.

In a third aspect, the invention provides a method of treating sepsis orchronic neuropathic pain comprising administration of a compound whichis an ester of 1,2,3-propanetriol and one or more fatty acids of atleast C11, wherein at least one fatty acid has a double bond at theC10-C11 position.

The method of the third aspect can be used to inhibit proinflammatorysignaling by activated macrophages and delay the development of sepsis,and this opens a time window to allow effective treatment of infectionunderlying the sepsis while preventing the organ damage that itproduces.

In a fourth aspect, the invention provides a method of treating sepsisand/or of preventing or treating post-sepsis syndrome, comprisingadministration of a fatty acid of at least C11 comprising a double bondbetween C10 and C11.

The method of the fourth aspect can be used to inhibit proinflammatorysignaling by activated macrophages and delay the development of sepsis,and this opens a time window to allow effective treatment of infectionunderlying the sepsis while preventing the organ damage that itproduces.

DESCRIPTION OF THE FIGURES

The invention is described herein with reference to the followingFigures.

FIGS. 1A and 1B show data obtained from Experiment 2 showing that M.vaccae and M. vaccae fraction 148.2 reduce bronchopulmonaryinflammation. Treatment with lipid fraction 148.2 of M. vaccae reducedthe severity of pulmonary allergic inflammation. FIG. 1A shows thatcompared to vehicle-treated control mice, subcutaneous treatment withwhole M. vaccae or lipid fraction 148.2 reduced the total number ofcells recovered in the bronchoalveolar lavage (BAL) fluid of allergicmice challenged with ovalbumin. Allergic mice were treated with vehicle(white bars) or with 0.1 mg of a heat-killed preparation of M. vaccae(Mva; black bars) or with 2 doses of lipid fraction 148.2 (grey bars).FIG. 1B shows that compared to vehicle-treated control mice, micetreated with M. vaccae or one of two doses of lipid fraction 148.2 hadreduced numbers of eosinophils (EOS) and macrophages (MΦ) recovered inthe BAL fluid of allergic mice challenged with ovalbumin. Data areexpressed as mean ±SEM of 6-8 mice per group. These results arerepresentative of two separate experiments. *p<0.05 relative tovehicle-treated controls as determined by ANOVA analysis. Abbreviations:EOS, eosinophils; MΦ, macrophages; NEUTS, neutrophils;

FIG. 2 shows data obtained from Experiment 3 showing that the synthetictriglyceride, 1,2,3-tri[Z-10-hexadecenoyl]glycerol, reducesbronchopulmonary inflammation. Treatment with a synthetic triglycerideoriginally isolated and purified from M. vaccae reduces the severity ofpulmonary allergic inflammation. Allergic mice were treated with vehicle(white bars), with 0.1 mg of heat-killed M. vaccae (black bars) or witha 5 μg dose of the synthetic triglyceride1,2,3-tri[Z-10-hexadecenoyl]glycerol (gray bars). Compared to controlmice, subcutaneous treatment with whole heat-killed M. vaccae reducedthe numbers of eosinophils (EOS) and macrophages (MΦ) recovered in thebronchoalveolar lavage (BAL) fluid of allergic mice challenged withovalbumin, while synthetic triglyceride1,2,3-tri[Z-10-hexadecenoyl]glycerol reduced the numbers of eosinophils(EOS) recovered in the BAL fluid of these mice. Data are expressed asmean ±SEM of 6-8 mice per group. These results are representative of twoseparate experiments. *p<0.05 when compared to treatment with vehicle asdetermined by ANOVA analysis. Abbreviations: EOS, eosinophils; MΦ,macrophages; NEUTS, neutrophils;

FIGS. 3A and 3B show data from Experiment 3 showing that the synthetictriglyceride, 1,2,3-tri[Z-10-hexadecenoyl]glycerol, has ananti-inflammatory effect on immune cells recovered by bronchoalveolarlavage (BAL) fluid and splenocytes. Treatment with a synthetic lipidoriginally obtained from M. vaccae altered the cytokine profile ofallergic mice. FIG. 3A shows the levels of interleukin (IL)-5 and IL-10measured in the bronchoalveolar lavage (BAL) fluid of allergic micetreated with buffer (white bars), with 0.1 mg of a heat-killedpreparation of M. vaccae (black bars) or with a 5 pg dose of thesynthetic triglyceride 1,2,3-tri[Z-10-hexadecenoyl]glycerol (gray bars).Treatment with 1,2,3-tri[Z-10- hexadecenoyl]glycerol reduced IL-5 levelsand increased IL-10 levels. FIG. 3B shows the levels of IL-5 and IL- 10measured in the supernatant of splenocytes stimulated in vitro withovalbumin. Splenocytes of mice treated with1,2,3-tri[Z-10-hexadecenoyl]glycerol produced less IL-5 and more IL-10.Data are expressed as mean ±SEM from pooled spleens of 6-8 mice pergroup. These results are representative of two separate experiments.

FIGS. 4A-4D show anti-inflammatory effects of 10(Z)-hexadecenoic acid infreshly-isolated murine peritoneal macrophages. Freshly isolated murineperitoneal macrophages were incubated for 1 h with 10(Z)-hexadecenoicacid (0.4 μM, 4 μM, 20 μM, 100 μM, 500 μM, 1000 μM), then challengedwith 1 μg/mL lipopolysaccharide (LPS). Cell supernatants were collectedat 6 h (FIG. 4A), 12 h (FIG. 4B), and 24 h (FIG. 4C) after LPSchallenge. Interleukin (IL)-6 concentrations in the supernatant weredetermined using ELISA and reported relative to media-only controls (n=6replicates, with each replicate using different freshly isolatedperitoneal macrophages; each sample was run in duplicate). FIG. 4D showsa control performed in the presence or absence of LPS. Shown are theeffects of incubation with 10(Z)-hexadecenoic acid for 1 h prior totreatment with 1 pg/mL LPS, replicating the effects shown in FIGS.4A-4C). Data for effects of 10(Z)-hexadecenoic acid in the absence ofLPS are not shown as all values were 0, indicating that10(Z)-hexadecenoic acid by, itself, has no detectable effect on IL-6secretion from macrophages.

FIG. 5 shows a macrophage cell viability assay. Sulforhodamine B (SRB)was used to assess cytotoxic effects of various concentrations of10(Z)-hexadecenoic acid (10 μM, 50 μM, 100 μM, 250 μM, 500 μM, 1000 μM)after 0, 6, 12, 24, 48, and 72 h of incubation with freshly isolatedmurine peritoneal macrophages. Percent control growth is expressed as %viability and is a ratio of the amount of growth that occurred withtreatment over the amount of growth that occurred in media. One hundredpercent indicates no differences in cell growth between treatment andmedia, whereas values below 100% indicate that growth was impaired withtreatment. Data are expressed as mean±SEM of 3-7 mice per condition.

FIG. 6 shows chemical structures associated with the synthesis of thetriacylglycerol.

FIG. 7 shows a diagrammatic illustration of six experimental designs, inwhich the following abbreviations are used: BAL, bronchoalveolar lavage;i.p., intraperitoneal; i.t., intratracheal; OVA, chicken egg ovalbumin;s.c., subcutaneous; DMEM, Dulbecco's Modified Eagle Medium; LPS,lipopolysaccharide; FBS, fetal bovine serum; DPBS, Dulbecco'sPhosphate-Buffered Saline.

DETAILED DESCRIPTION

The present invention provides lipid compounds or fatty acids for thetreatment of sepsis and the prevention or treatment of post-sepsissyndrome. More specifically the lipid compounds for the treatment ofsepsis are fatty acids, or mono-, di- and tri-esters of the fatty acidand 1,2,3-propanetriol. The lipids or fatty acids can be synthesized invitro. Alternatively, the lipid compound or fatty acid may be derived orextracted from Mycobacterium vaccae. M. vaccae is sometimes referred toas DAR-901 and a suitably exemplary strain is deposited under the NCTCaccession number NCTC 11659.

In another aspect of the invention, the lipid compound or fatty acid maybe derived or extracted from a Mycobacterium selected from the listincluding M. vaccae, M. thermoresistibile, M. flavescens, M. duvalii, M.phlei, M. obuense, M. parafortuitum, M. sphagni, M. aichiense, M.rhodesiae, M. neoaurum, M. chubuense, M. tokaiense, M. komossense, M.aurum, M. indicus pranii, M. tuberculosis, M. microti; M. africanum; M.kansasii, M. marinum; M. simiae; M. gastri; M. nonchromogenicum; M.terrae; M. triviale; M. gordonae; M. scrofulaceum; M. paraffinicum; M.intracellulare; M. avium; M. xenopi; M. ulcerans; M. diernhoferi, M.smegmatis; M. thamnopheos; M. flavescens; M. fortuitum; M. peregrinum;M. chelonei; M. paratuberculosis; M. leprae; M. lepraemurium andcombinations thereof.

The term “sepsis” in the context of the present invention is understoodto mean a life-threatening condition that arises when the body'sresponse to infection injures its own tissues and organs. Common signsand symptoms include fever, increased heart rate, increased breathingrate and confusion. There may also be symptoms related to a specificinfection, such as a cough with pneumonia, or painful urination with akidney infection. In the very young, old, and people with weakenedimmune systems, there may be no symptoms of a specific infection andbody temperature may be normal or lower than normal rather than high.Severe sepsis is sepsis causing poor organ function or insufficientblood flow. Insufficient blood flow may be evident by low bloodpressure, elevated blood lactate or low urine output. Septic shock islow blood pressure due to sepsis that does not improve after reasonableamounts of intravenous fluids are given.

“Post-sepsis syndrome”, or “PSS”, in the context of the presentinvention is understood to be a multi-faceted condition that can afflictup to 50% of subjects who recover from a bout of sepsis. The disordermanifests with a number of distinct, overlapping symptoms, including,but not limited to insomnia, vivid hallucinations, panic attacks, pain,particularly in muscles and joints, extreme fatigue, poor concentrationand reduced cognitive function. PSS is thought to arise by a variety ofmechanisms, with localised pain and neurological defects are thought toarise at least in part due to organ damage and/or failure brought aboutby the inflammatory response underlying sepsis.

Thus, in an embodiment of the invention, the compounds, fatty acids, andcompositions disclosed herein in may be used to treat PSS and symptomsassociated therewith.

As disclosed herein, “treatment” of PSS encompasses the prevention,ablation or partial reduction of symptoms of PSS. In one embodiment, thecompounds, fatty acids, and compositions disclosed herein prevent,ablate or partially reduce all or a number of the following symptoms:insomnia, vivid hallucinations, panic attacks, pain, particularly inmuscles and joints, extreme fatigue, poor concentration and reducedcognitive function. In one embodiment, the compounds, fatty acids, andcompositions disclosed herein prevent, ablate or partially reducePSS-associated pain. In one embodiment, PSS-associated pain is chronicneuropathic pain. In another embodiment, the compounds, fatty acids, andcompositions disclosed herein prevent, ablate or partially reducePSS-associated neurological defects.

It is envisaged that the improvement, reduction or elimination of thesesymptoms is not restricted to patients in the post-sepsis phase. The useof the compounds, fatty acids, and compositions in the methods disclosedherein can also alleviate these symptoms experienced by the patient whenadministered during an on-going bout of sepsis.

The present inventors have found that compounds of the invention preventlipopolysaccharide-stimulated secretion of the proinflammatory cytokineIL-6 from freshly isolated peritoneal macrophages. The present inventorshave also found that such compounds down-regulate expression ofproinflammatory cytokine and chemokine ligand mRNAs in activatedmacrophages and inhibit the proinflammatory signaling of activatedmacrophages in vitro,. This can be measured using RNAseq analysis offreshly isolated murine peritoneal macrophages stimulated with LPS, inthe presence or absence of 10(Z)-hexadecenoic acid. Pathway analysispredicted that the lipid activates peroxisome proliferator-activatedreceptor signaling. Compounds of the invention are therefore useful ininhibiting the proinflammatory cascade that contributes to the pathologyof sepsis.

In a first aspect the invention provides a compound for use in thetreatment of sepsis which is an ester of 1,2,3-propanetriol with one ormore fatty acids of at least C11, wherein at least one fatty acid has adouble bond at the C10-C11 position. The compound will have the generalformula:

where R₁, R₂ and R₃ may be the same or different and are either H or aresidue of a fatty acid, wherein at least one of R₁, R₂ and R₃ is otherthan H, and at least one of the fatty acids is at least C11 and has adouble bond at the C10-C11 position.

The compound for use in the treatment of sepsis is either a mono-, di-or tri-ester. Preferably the compound for use in the treatment of sepsisis a tri-ester. When the compound for use in the treatment of sepsis isa mono-ester, esterification may occur at the 1- or 2-position. When thecompound for use in the treatment of sepsis is a mono-ester preferablyesterification occurs at the 1-position. When the compound for use inthe treatment of sepsis is a di-ester, esterification may occur at the1- and 2-positions or at the 1- and 3-positions. When the compound foruse in the treatment of sepsis is a di-ester preferably esterificationoccurs at the 1- and 3-positions.

When the compound for use in the treatment of sepsis is a di-ester thefatty acids may be the same or the fatty acids may be different.Preferably when the compound for use in the treatment of sepsis is adi-ester the fatty acids are the same.

When the compound for use in the treatment of sepsis is a tri-ester thefatty acids may be the same or the fatty acids may be different.Preferably when the compound for use in the treatment of sepsis is atri-ester the fatty acids are the same.

At least one fatty acid of the compound for use in the treatment ofsepsis has a double bond at position C10-C11. The double bond may be cisor trans. In one embodiment, the double bond is cis. In anotherembodiment, the double bond is trans.

The fatty acid is suitably a C₁₁ to C₂₄ fatty acid, e.g. C₁₁, C₁₂, C₁₄,C₁₆, C₁₈, C₂₀, C₂₂ or C₂₄, preferably a C₁₁ to C₂₀ fatty acid, morepreferably a C₁₁ to C₁₆ fatty acid. In one embodiment at least one fattyacid with a C10-C11 double bond is a C16 fatty acid.

The compound for use in the treatment of sepsis is preferably atri-ester of 1,2,3-propanetriol in which at least one of the fatty acidsis 10(Z)-hexadecenoic acid.

In a second aspect the invention provides a fatty acid of at least C11comprising a double bond at the C10-C11 position for use in thetreatment of sepsis. The C10-C11 double bond may be cis or trans. In oneembodiment, the double bond is cis. In another embodiment, the doublebond is trans. The fatty acid may contain more than one double bond.

The fatty acid may be any C₁₁ to C₂₄ fatty acid, e.g. C₁₁, C₁₂, C₁₄,C₁₆, C₁₈, C₂₀, C₂₂ or C₂₄, preferably a C₁₁ to C₂₀ fatty acid, morepreferably a C₁₁ to C₁₆ fatty acid. In one embodiment the fatty acidwith a C10-C11 double bond is a C₁₆ fatty acid.

In a particular embodiment the C₁₁ to C₂₄ fatty acid is10(Z)-hexadecenoic acid.

In certain embodiments the invention provides a composition comprisingthe compound of the first aspect or the fatty acid of the second aspectin conjunction with an antibacterial, anti-fungal, or anti-viralmolecule that eradicates the infectious agent causing sepsis.

In other embodiments the compound of the first aspect or the fatty acidof the second aspect are for separate, sequential, or simultaneousadministration with an antibacterial, anti-fungal, or anti-viralmolecule that eradicates an infectious agent causing sepsis, for use inthe treatment of sepsis.

The invention also provides the use of the compounds as defined above inthe preparation of a medicament for the treatment of sepsis. Theinventors have found that the free fatty acid alone is also effectiveand the invention further provides the use of a C₁₁-C₂₄ fatty acidhaving at least one C10-C11 double bond in the preparation of amedicament for the treatment of sepsis. The fatty acid is preferablyC₁₁-C₁₆, and more preferably is 10(Z)-hexadecenoic acid.

The compounds, fatty acids and compositions of the invention as definedabove are administered by the parenteral route. In particularembodiments, they are administered by subcutaneous, intradermal,subdermal, intraperitoneal, intravenous or intravesicular injection. Inpreferred embodiments they are administered by intravenous injection.

The compounds of the invention or the free fatty acids as defined abovemay be used in the preparation of a medicament. The medicament mayfurther comprise standard pharmaceutically acceptable carriers and/orexcipients as is routine in the pharmaceutical art. For example, thecompound of the invention or the defined free fatty acid may be put intosuspension in, for example, a physiological buffer, isotonic saline orwater by physical disruption such as ultrasound. Alternatively it may beput into suspension by ultrasound in the presence of a stable carrierprotein, for example lipid-free human serum albumin, to which the lipidand/or glycolipid will bind, providing a stable solution.

Alternatively the compounds of the invention or the free fatty acids asdefined above may be formulated as slow release pellets followingcombination with a suitable carrier molecule, for example cholesterol. Asuitable carbohydrate that is linked to a lipid or glycolipid may beformulated in the same way as a lipid and/or glycolipid. A suitablecarbohydrate not linked to a lipid or glycolipid may be dissolved in,for example, physiological saline or water for injection. The exactnature of a formulation will depend upon several factors including theparticular substance to be administered and the desired route ofadministration. Suitable types of formulation are fully described inRemington's Pharmaceutical Sciences, Mack Publishing Company, EasternPennsylvania, 17.sup.th Ed. 1985, the disclosure of which is includedherein of its entirety by way of reference.

The pharmaceutical composition comprising compounds of the invention orthe free fatty acids as defined above may also contain furtheringredients such as adjuvants, preservatives, stabilisers etc. It mayfurther comprise other therapeutic agents. It may be supplied in sterileand pyrogen-free form, for example as an injectable liquid; in sterilefreeze-dried form which is reconstituted prior to use; or as sterileslow-release pellets. The pharmaceutical composition may be supplied asan isotonic liquid. It may be supplied in unit dosage form.

In a third aspect the invention provides a method of treating sepsiscomprising administration of a compound which is an ester of1,2,3-propanetriol and one or more fatty acids of at least C11, whereinat least one fatty acid has at least one double bond, at the C10-C11position, to a patient in need of such treatment.

The compound can be a mono-, di- or tri-ester, but preferably it is atri-ester. When the compound is a mono-ester, esterification may occurat the 1- or 2-position, but preferably esterification occurs at the1-position. When the compound is a di-ester, esterification may occur atthe 1- and 2-positions or at the 1- and 3-positions, but preferablyesterification occurs at the 1- and 3-positions.

When the compound is a di-ester or a tri-ester the fatty acids may bethe same or the fatty acids may be different, but preferably the fattyacids are the same.

At least one fatty acid of the compound has at least one double bond, atposition C10-C11. The double bond may be cis or trans. In oneembodiment, the double bond is cis. In another embodiment, the doublebond is trans. The fatty acid may comprise a single double bond, at theC10-C11 position.

The fatty acid is suitably a C₁₁ to C₂₄ fatty acid, e.g. C₁₁, C₁₂, C₁₄,C₁₆, C₁₈, C₂₀, C₂₂ or C₂₄, preferably a C₁₁ to C₂₀ fatty acid, morepreferably a C₁₁ to C₁₆ fatty acid. In one embodiment the at least onefatty acid with a C10-C11 double bond is a C₁₆ fatty acid.

The compound is preferably a tri-ester of 1,2,3-propanetriol in which atleast one of the fatty acids is 10(Z)-hexadecenoic acid.

In preferred embodiments the method further comprises the separate,sequential, or simultaneous administration of an antibacterial,anti-fungal, or anti-viral molecule known to eradicate the infectiveagent causing sepsis.

In a fourth aspect the invention provides a method of treating sepsiscomprising administration of a fatty acid of at least C11 comprising adouble bond between C10 and C11 to a patient in need of such treatment.

The fatty acid may be any C₁₁ to C₂₄ fatty acid, e.g. C₁₁, C₁₂, C₁₄,C₁₆, C₁₈, C₂₀, C₂₂ or C₂₄, preferably a C₁₁ to C₂₀ fatty acid, morepreferably a C₁₁ to C₁₆ fatty acid. In one embodiment the fatty acidwith a C10-C11 double bond is a C₁₆ fatty acid.

In a particular embodiment the fatty acid is 10(Z)-hexadecenoic acid.

In preferred embodiments the method further comprises the separate,sequential, or simultaneous administration of an antibacterial,anti-fungal, or anti-viral molecule known to eradicate the infectiousagent causing sepsis to a patient in need of such treatment.

In the methods of the invention the compounds, fatty acids andcompositions as defined above are administered by the parenteral route.In particular embodiments of the method they are administered bysubcutaneous, intradermal, subdermal, intraperitoneal, intravenous orintravesicular injection. In preferred embodiments they are administeredby intravenous injection.

In another embodiment, the compounds, fatty acids and compositions asdefined above are administered via the enteral route, preferably byintratracheal administration. In one embodiment, the compound, fattyacids and compositions are administered via an inhaler.

A therapeutically effective amount of the compound of the invention orthe free fatty acids as defined above is administered to a patient. Thedose may be determined according to various parameters, especiallyaccording to the substance used; the age, weight and condition of thepatient to be treated; the route of administration; and the requiredregimen. A physician will be able to determine the required route ofadministration and dosage for any particular patient. Multiple doses maybe given. A typical individual dose is from about 0.005 to 1.0 mg/kg,preferably from about 0.01 to 0.5 mg/kg, more preferably from about 0.03to 0.3 mg/kg, according to the activity of the specific lipidpreparation, the age, weight and condition of the subject to be treated,the type and severity of the condition and the frequency and route ofadministration.

For the purposes of the present invention, the lipids derived fromMycobacteria can be extracted by a number of alternative processes wellknown in the art. For example, cells containing the lipid of interestcan be incubated with an extraction media that promotes dissolution ofthe lipids from the other components of the cell. Generally, suitableextraction media will comprise a mixture of organic and aqueous solventsthat when incubated for sufficient time separate into immiscible layersbased on a gradation of hydrophobicity. After a period of incubation ofthe cells in the extraction media under conditions that favour thedissolution of cells, the extraction media can be allowed to settle intothe individual immiscible layers (or aqueous phases), each of which willcontain a different cellular faction or, more specifically, a differentlipid fraction, with the most apolar lipids existing in the mosthydrophobic component of the extraction media. The different aqueousphases can then be individually aspirated to separate the various lipidfractions. Further separation of the lipids within each layer ispossible; by rehydrating the dried lipid films in a secondary extractionmedia. One particular non-limiting example of an extraction mediasuitable for the primary extraction of lipids comprises a mixture ofpetroleum ether, methanol and 0.3% aqueous sodium chloride solution.Other suitable media for primary and secondary extractions will be wellknown to the skilled artisan. Alternatively, or in combination with theabove, lipids may be extracted or purified from the milieu usinggradient liquid chromatography in a mixture of suitable organicsolvents, such as increasing amounts of methanol in chloroform, or bythin-film chromatography. The identity of the extracted lipid can bedetermined using any number of analytical techniques, including, but notlimited to, mass spectrometry, infrared spectroscopy and 1D-NMR.

Compounds for use in the treatment of sepsis of the invention may besynthesised by a process which is defined in the examples accompanied bya schematic illustration in FIG. 6. More generally, however, the skilledperson would appreciate that the starting materials of the chemicalsynthesis could be modified in order to control or modify the length ofthe fatty acid chain. The skilled person would also acknowledge that thereaction times, conditions, temperature, pressure, reactantconcentrations, and yields resulting from each step of the synthesis mayvary according to the starting materials used. Additional steps couldalso be incorporated in order to introduce additional double bonds intothe fatty acid molecule. The optimisation of each step within thesynthetic route when using different starting materials is well withinthe purview of the skilled person. Fundamentally, modifying the startingmaterials simply requires trial and error in order to achieve effectivesynthesis of the fatty acid molecule, or triacylglycerol esters thereof,of varying chain length that comprises at least a double bond atpositions C10 to C11.

Throughout the present specification and the accompanying claims thewords “comprise” and “include” and variations such as “comprises”,“comprising”, “includes” and “including” are to be interpretedinclusively. That is, these words are intended to convey the possibleinclusion of other elements or integers not specifically recited, wherethe context allows.

EXAMPLES

The fractionation of M. vaccae NCTC 11659 with a number of solventsresulted in the isolation of several lipid fractions with differentcharacteristics. Preliminary work identified lipids present in theaqueous methanol fraction, such as phospholipids, polar and neutralglycolipids and glycosylphosphatidylinositols, but not those in thepetrol fraction, such as phthiocerols, dimicocerosates and mycolicacids, to be of considerable interest.

Pretreatment with an Aqueous Methanol Extraction of M. vaccae ReducesAllergic Pulmonary Inflammation

Upon further fractionation of the aqueous methanol fraction, thephospholipid, polar glycolipid and glycosyl phosphatidylinositolcomponents (fraction 147) were separated from the neutral glycolipidcomponents (fraction 148).

In Experiment 1, the immunomodulatory properties of the two fractionswere assessed in vitro using Thp1 cells, a human monocytic cell line,exposed overnight to these preparations (for experimental timeline, seeFIG. 7, Experiment 1). It was found that Thp1 cells stimulated withfraction 147 preferentially produce IL-12p40. In contrast whenstimulated with fraction 148 they showed reduced IL-12p40 secretion andan increase in IL-10 levels.

In Experiment 2 mice were treated, subsequently rendered allergic byimmunization with ovalbumin and alum on Days 0 and 12, with either aheat-killed preparation of M. vaccae (NCTC 11659; 0.1 mg, s.c.) orfraction 147 and 148, (5 μg; s.c.) on Day −21 and determined theirrespective potential in limiting allergic pulmonary inflammation,measured on Day 22 following intratracheal (i.t.) ovalbumin challenge onDays 19 and 21 (for experimental timeline, see FIG. 7, Experiment 2).

Pulmonary allergic inflammation is associated with a large influx ofcells, particularly eosinophils, in the airway. We found that micetreated with either a heat-killed preparation of M. vaccae NCTC 11659 orfraction 148 showed a significant reduction in both the total cellularinfiltrate and in the number of eosinophils recovered in thebronchoalveolar lavage (BAL) fluid (Table 1).

The decrease in eosinophils was not associated with an increase in otherinflammatory cells. In fact, macrophages (p<0.05) were also reduced atthe site of inflammation, while there were no effects on numbers ofneutrophils (Table 1).

This reduction in disease severity was not observed in allergic micetreated with fraction 147. These results suggest that, not only dofractions 147 and 148 have different immunological properties, but alsothat fraction 148 prevents pulmonary allergic inflammation in a mousemodel.

TABLE 1 Characterization of the cellular infiltrate in the lungs ofallergic mice challenged intratracheally with ovalbumin in Experiment 2.Cell × 10⁵/ml M. vaccae Vehicle NCTC 11659 Fraction 147 Fraction 148Total cellular 2.14 ± 0.2 0.65 ± 0.1* 1.73 ± 0.36 0.55 ± 0.1* infiltrateEosinophils 0.41 ± 0.1 0.17 ± 0.01* 0.49 ± 0.2 0.12 ± 0.01* Neutrophils0.12 ± 0.04 0.03 ± 0.02 0.07 ± 0.02 0.02 ± 0.02 Macrophages 1.57 ± 0.10.45 ± 0.1* 1.15 ± 0.2 0.42 ± 0.1* *denotes p < 0.05 inBonferroni-corrected ANOVA compared with vehicle-treated,ovalbumin-challenged mice. These results are representative of at leasttwo separate experiments (n = 8 per group).

Specific Fractions of the Mycobacterial Extract are Anti-Inflammatory InVitro

Fraction 148 was further fractionated by column chromatography using achloroform-methanol gradient. Based on previous results we screenedseven subfractions of fraction 148 using Thp1 cells for a cytokineprofile associated with low IL-12p40 and increased IL-10 levels.Following these criteria, three subfractions were identified as havinginteresting features, and fraction 148.2 was used in furtherexperiments.

In Experiment 3 (for experimental timeline, see FIG. 7, Experiment 3) itwas demonstrated that mice treated with either M. vaccae NCTC 11659 (0.1mg, s.c.) or two different doses of fraction 148.2 (5 μg and 1 μg, s.c.)showed a significant reduction in both the total cellular infiltrateand/or the number of eosinophils or macrophages recovered in the BALfluid (FIGS. 1A and 1B).

Additionally, in Experiment 4 (for experimental timeline, see FIG. 7,Experiment 4), the effects of M. vaccae NCTC 11659 (0.1 mg, s.c.) andfraction 148.2 (5 μg or 1 μg, s.c.) on IL-10 secretion from splenocytesstimulated with allergen ex vivo were studied. Splenocytes from micetreated with M. vaccae NCTC 11659 and mice treated with fraction 148.2produced increased levels of IL-10 following in vitro allergen(ovalbumin) stimulation (M. vaccae NCTC 11659, 234±47 pg/ml; 1 μgfraction 148.2, 227±20 pg/ml; 5 μg fraction 148.2, 291±16 pg/ml),compared to levels from splenocytes of mice treated with vehicle alone(179±37 pg/ml). These results suggest that components of fraction 148.2maintain the induction, in vitro, of a cytokine profile associated withprevention of pulmonary allergic inflammation in a mouse model.

The Anti-Inflammatory Component of the Fraction is a triglyceride

By performing a series of analytic chemistry experiments including highresolution mass spectrometry (HRMS), 1-dimensional (1D) (¹H and¹³C) andtwo-dimensional (2D) (correlation spectroscopy (COSY) and ¹H/¹³Cheteronuclear multiple bond correlation (HMBC)) nuclear magneticresonance (NMR) spectroscopy, and gas chromatography-mass spectrometry(GC-MS) analyses, it was concluded that fraction 148.2 is atriacylglycerol. From the ¹H signals a glycerol backbone at 4.20 ppm(4H) and 5.25 ppm (1H) was identified. A complex signal at 5.32 ppm(3.3H, CH═CH), 2.3 ppm (6H, CO—CH₂), 2.0 ppm (8H, CH₂CH═CHCH₂) and1.6ppm (8H, CO—CH₂CH₂) was also obtained. Electro-spray massspectrometry analysis of the triacylglycerol yielded the sodiatedmolecular ion at a mass-to-charge ratio (m/z) 823. Further analysis offatty acid characteristics revealed that fraction 148.2 containedpredominantly C16 and C18:1 and a smaller proportion of C18 and C16:1.The position of the double bond was determined. The connectivity patternwas consistent with a cis double bond for C18:1 at position 9,10 and,surprisingly, at position 10,11 for C16:1. The latter is a ratherunusual lipid and appears to be restricted to Mycobacteria spp. (seecompound (8) in FIG. 6), although lactobacilli have been shown to havecapacity to synthesize, from γ-linolenic acid, C18:1 fatty acids with adouble bond at position 10,11 (Ogawa, J. et al. (2005) J. Biosci.Bioeng. 100:355-364).

The triglyceride, 1,2,3-tri[Z-10-hexadecenoyl]glycerol, has TherapeuticPotential in a Mouse Model of Pulmonary Allergic Inflammation

Whether treatment with this triacylglycerol alone retains thetherapeutic activity initially observed for treatment with whole M.vaccae NCTC 11659 was investigated in Experiment 5. To this end, theC16:1 triacylglycerol with a double bond at position 10,11 of each acylchain (hereafter referred to as 1,2,3-tri[Z-10-hexadecenoyl]glycerol)was synthesized and its therapeutic potential in a mouse model ofallergic pulmonary inflammation was determined (for experimentaltimeline, see FIG. 7, Experiment 5).

It was found that mice treated with either M. vaccae NCTC 11659 (0.1 mg,s.c.) or 1,2,3-tri[Z-10-hexadecenoyl]glycerol (5 μg, s.c.) showed areduction in both the total cellular infiltrate and in the number ofeosinophils recovered in the BAL fluid (FIG. 2). Once again thereduction in eosinophilia was not accompanied by an increase inType1-mediated inflammation as the numbers of macrophages were notincreased. The reduction in the number of eosinophils was comparable tothat seen following treatment with heat-killed whole cell M. vaccae NCTC11659 (FIG. 2).

Additionally, a decrease in IL-5 concentrations and an increase in IL-10concentrations in the BAL fluid of mice treated with1,2,3-tri[Z-10-hexadecenoyl]glycerol compared to those measured in theBAL fluid of mice treated with vehicle were observed (FIG. 3A). Levelsof IFN-γ were below detection in the BAL fluid of allergic miceregardless of treatment. Cytokine levels were measured in thesupernatant of in vitro splenocyte culture following allergenstimulation. An increase in IL-10 and a decrease in IL-5 levels in thesupernatant of splenocytes of mice treatedwith1,2,3-tri[Z-10-hexadecenoyl]glycerol were found compared to those inthe supernatant of splenocytes of mice treated with buffer (FIG. 3B).These results are the first to show therapeutic activity of a syntheticlipid, 1,2,3-tri[Z-10-hexadecenoyl]glycerol, of M. vaccae NCTC 11659 ina mouse model of pulmonary allergic inflammation.

The free fatty acid, 10(Z)-hexadecenoic acid, was also synthesized inorder to determine if the triglyceride structure was necessary fortherapeutic function of 1,2,3-tri[Z-10-hexadecenoyl]glycerol. This wastested in Experiment 6 (for experimental timeline, see FIG. 7,Experiment 6).

In the mouse model of allergic pulmonary inflammation, the free fattyacid provided a significant reduction of both eosinophilia and totalcellular infiltrate. Furthermore, 10(Z)-hexadecenoic acid treatment didnot induce increases in macrophages or neutrophils. As was seen for1,2,3-tri[Z-10-hexadecenoyl]glycerol, a decrease in IL-5 concentrationsand an increase in IL-10 concentrations in the BAL fluid of10(Z)-hexadecenoic acid-treated mice was observed. From these results,it was concluded the anti-inflammatory effects of1,2,3-tri[Z-10-hexadecenoyl]glycerol are not due to the molecule'smultivalency, but rather they are due to its acyl chains.

10(Z)-hexadecenoic Acid Prevents LPS-Induced Secretion of IL-6 inMacrophages

Experiment 7 was carried out to resolve if 10(Z)-hexadecenoic acid has adirect effect on immune cells. Macrophages were selected for this studyon account of their ubiquity and diverse role in immune processes. Tosimulate inflammation, freshly isolated mouse peritoneal macrophageswere challenged with lipopolysaccharide (LPS; 1 μg/m L) ex vivo.

Macrophages that were cultured in the presence of 10(Z)-hexadecenoicacid for 1 h prior to LPS treatment secreted less IL-6 compared tomacrophages cultured with media alone (FIGS. 4A-4C). This difference wasobservable as early as 6 h after LPS challenge, and was sustained for atleast 24h. The effect also appeared to be concentration and timedependent. The lowest concentration of 0.4 μM was ineffective at 6 h,but reduced IL-6 secretion to 40% of control levels at 24 h. This timeand concentration dependence may indicate that a receptor-mediatedtranscriptional change is occurring. In contrast to the effects of10(Z)-hexadecenoic acid on LPS-induced IL-6 secretion, it had no effectof IL-6 secretion by itself (FIG. 4D). A transcriptional change wasconfirmed by measurement of pro-inflammatory cytokine and chemokineligand mRNA levels in macrophages pre-treated with 10(Z)-hexadecenoicacid for 1 h prior to LPS treatment (Table 2).

Cell viability was measured to dispel the possibility that senescence orcell death was contributing to reduced IL-6 secretion. Using a highconcentration (1 mM) of 10(Z)-hexadecenoic acid, less than 40% ofmacrophages were viable at most time points. However, macrophagescultured with all other concentrations of 10(Z)-hexadecenoic acid wereas viable as or more viable than media controls (FIG. 5).

TABLE 2 Proinflammatory cytokine and chemokine ligand mRNAsdownregulated by preincubation of freshly isolated murine peritonealmacrophages with a synthetic, M. vaccae-derived fatty acid,10(Z)-hexadecenoic acid, prior to stimulation with lipopolysaccharide.Mean #Reads Mean s148.2 + Log2 #Reads LPS (n = Fold- Fold- Adjusted GeneLPS (n = 3) 3) Effect change change p value p value Il6 1302 305 ↓ 0.23−2.09 4.19E−23  4.37E−19 Il1b 15,957 5349 ↓ 0.34 −2.16 4.03E−15 6.48E−12 Ccl7 1182 441 ↓ 0.37 −1.42 3.37E−12 3.35E−9 Ccl2 5656 2234 ↓0.39 −1.34 5.18E−12 4.91E−9 Il12a 714 268 ↓ 0.37 −1.42 5.70E−11 4.57E−8Ccl22 327 117 ↓ 0.36 −1.48 5.15E−10 3.26E−7 Ccl6 810 368 ↓ 0.45 −1.145.31E−8 2.36E−5 Tnf 2098 1023 ↓ 0.49 −1.04 2.95E−7 1.04E−4 Ccl17 24 3 ↓0.12 −3.08 3.97E−7 1.34E−4 Il1a 16533 8500 ↓ 0.51 −0.96 1.02E−6 3.19E−4Ccl4 603 305 ↓ 0.51 −0.98 3.75E−6 1.02E−3 Il11 26 5 ↓ 0.20 −2.35 1.59E−5 3.7E−3 Cxcl2¹ 4726 2680 ↓ 0.57 −0.82 3.01E−5  6.4E−3 Ccl3 6693 4067 ↓0.61 −0.72 1.99E−4  2.9E−2 ¹Cxcl2 is a functional homologue of humanIL-8

Experimental Materials and Methods

Animals

For studies involving isolation of bacterially-derived small molecules,and in vivo studies, adult female BALB/c mice, 6-8 weeks old, (Harlan,Abingdon, UK) were housed under standard conditions with food and wateravailable ad libitum. For studies involving ex vivo studies of freshlyisolated peritoneal macrophages, adult male BALB/c mice (BALB/cAnHsd;Cat. No. 047; Harlan, Indianapolis, Ind., USA), 6-8 weeks old, were usedand housed under standard conditions with food and water available adlibitum.

For studies involving isolation of bacterially-derived small molecules,and in vivo studies, all experimental protocols complied with the HomeOffice 1986 Animals Scientific Act. For studies involving ex vivostudies of freshly isolated peritoneal macrophages, all experimentalprotocols were consistent with the National Institutes of Health Guidefor the Care and Use of Laboratory Animals, Eighth Edition (The NationalAcademies Press, 2011) and the Institutional Animal Care and UseCommittee at the University of Colorado Boulder approved all procedures.All possible efforts were made to minimize the number of animals usedand their suffering.

Mycobacterium vaccae

Isolation of bacterially-derived small molecules from M. vaccae wasconducted using sterile paste derived from freshly grown stocks. For invivo studies, sterile vials of heat-killed whole cell M. vaccaesuspension (10 mg/ml; strain NCTC 11659) were supplied by SR Pharma(London, UK). For studies involving ex vivo studies of freshly isolatedperitoneal macrophages, the same material was provided by BioElpida(Lyon, France; batch ENG#1).

Isolation of Lipids

Wet cells (200 g of paste of sterile heat-killed whole cell M. vaccae)were extracted using 440 mL of petroleum ether, 400 mL of methanol, and40 mL 0.3% aqueous sodium chloride overnight with gentle agitation. Themixture was then left to stand and the upper organic petroleum-ethersupernatant fraction was separated by careful aspiration. The loweraqueous phase was extracted again using petroleum ether (400 mL) asdescribed above. The petroleum-ether extracts were combined and dried toyield the apolar lipids. The lower aqueous phase was then extractedusing chloroform/methanol/water (90:100:30; 520 mL) with gentleagitation, overnight. The resulting lipid extract was separated byvacuum filtration and the residual biomass extracted usingchloroform/methanol/water (50:100:40; 170 mL) overnight with gentleagitation twice. The three polar lipid extractions were combined andchloroform (290 mL) and 0.3% aqueous sodium chloride (290 mL) was added.The entire mixture was briefly shaken, allowed to settle and the upperphase was carefully removed and discarded. The lower organic layer wasdried to yield the polar lipids. The polar lipids were resuspended in aminimum volume of chloroform (20 mL) and added to chilled acetone (1.5L) and left at 4° C., overnight. The resulting precipitate (lipidfraction 147) was separated by centrifugation from the acetone solublelipids (220 mg) designated lipid fraction 148. Fraction 148 was furtherfractioned using column chromatography using increasing amounts ofmethanol in chloroform to afford seven lipidic fractions. These werescreened for their immunomodulatory potential as described below. Whilea number of fractions were deemed interesting, fraction 148.2 (82 mg)was further analyzed. The resulting fraction was deemed pure bythin-layer chromatography (TLC) using chloroform as an eluant followingcharring with a heat gun after spraying with 5%ethanolicmolybdophosphoric acid. Through a combination of highresolution mass spectrometry (HRMS), 1-dimensional (1D) (¹H and¹³C), andtwo-dimensional (2D) (correlation spectroscopy (COSY) and ¹H/¹³Cheteronuclear multiple bond correlation (HMBC)) nuclear magneticresonance (NMR) spectroscopy, and gas chromatography-mass spectrometry(GC-MS) analyses, the structure of the triglyceride was completelydetermined.

Synthesis of triacylglycerol

The synthesis of triacylglycerol was based on the method of Besra et al.(Besra, G. S. et al. (1993) Chem. Phys. Lipids 66:23-34). Briefly, theacetylenic carboxylic acid (1) and trimethylsilyl chloride (0.1equivalent) in anhydrous methanol were mixed at room temperature for 12hours. The reaction was evaporated to dryness to yield the pure methylester product (2) as confirmed by thin layer chromatography (TLC) and¹H/¹³C-NMR analysis and was used directly in the next step withoutfurther purification. The carboxylic acid methyl ester (2) was dissolvedin diethyl ether and 2 equivalents of lithium aluminium hydride wereadded and the reaction was stirred at room temperature for 4 hours. Thereaction was quenched with glacial acetic acid and the acetylenicalcohol product (3) was extracted with diethyl ether and water. Theethereal layer was recovered and washed with water and then brine, thenconcentrated to dryness. To a solution of the acetylenic alcohol (3) (1equivalent) in hexamethylphosphoramide (HMPA), n-butyl lithium (2equivalents) was added at 0° C. under nitrogen over a period of 30 min.The reaction was stirred at 0° C. for 20 min. 1-iodopentane (1.4equivalent) was added and the reaction mixture was left to warm toambient temperature and stirred for 20 hours. The reaction was quenchedwith the addition of saturated aqueous ammonium chloride and the product(4) was extracted with diethyl-ether. The product (4) was concentratedand purified by column chromatography using a petroleum ether-ethylacetate gradient, monitored by TLC and characterized by ¹H/¹³C-NMR. Asuspension of Lindlar's catalyst in dry benzene was saturated withhydrogen gas and cooled to 10° C. Then a solution of (4) in benzene andquinoline was added under a stream of nitrogen. The reaction mixture wasstirred for 1 hour at 10° C. The reaction mixture was filtered,concentrated and the product (5) was purified by column chromatographyusing a petroleum ether-ethyl acetate gradient, monitored by TLC andcharacterized by ¹H/¹³C-NMR. A solution of (5) in dichloromethane (1volume) was added to a stirring solution of pyridinium dichromate (4equivalents) in dimethylformamide (DMF, 10 volumes). The reactionmixture was stirred for 2 days at room temperature. Water was added andthe product (6) was extracted into dichloromethane, washed with brineand concentrated. The product (6) was purified by column chromatographyand characterized by MS and ¹H/¹³C-NMR. The starting acid (6) wasdissolved in dichloromethane/DMF and oxalyl chloride was added; thereaction mixture was then stirred at room temperature for 1 hour. Thereaction mixture was evaporated and the crude acid chloride (7) was usedin the next step. Glycerol (1 equivalent) in pyridine was added to theacid chloride (7) (3.3. equivalents) and the reaction mixture was leftto stir overnight. Dichloromethane and water were added to the reactionmixture and the product was recovered in the organic layer andconcentrated. The synthetic triacylglycerol was purified by columnchromatography using increasing methanol in chloroform, monitored by TLCand characterized by MS, and ¹H/¹³C-NMR analyses. All indicatedstructures are shown in FIG. 6.

Thp 1 Cell Assay

Thp1 cells were used as an immunological screen to characterizeproperties of the isolated lipid fractions of M. vaccae in terms ofIL-12p40 and IL-10 secretion. Thp1 cells (ATCC, Teddington, UK) weredifferentiated overnight with 1.2% DMSO (Cat. No. D-5879; Sigma-Aldrich,Gillingham, UK) in culture media containing RPMI 1640 medium containing20 mM Hepes buffer without L-glutamine (Cat. No. 42402-016; Gibco BRL,Grand Island, N.Y., USA), containing 10% fetal calf serum (Cat. No.10106-169; Gibco BRLBRL), and 2 mM L-glutamine (Cat. no. 25030-024;Gibco BRLBRL). Cells were washed, counted and resuspended at aconcentration of 2×10⁶/ml. Cells were stimulated in vitro with isolatedlipid fractions of M. vaccae at 37° C. and 5% CO₂. Supernatants werecollected after 24 or 48 hours and cytokine concentrations were measuredusing commercially available ELISA kits (R&D Systems, Abingdon, UK). Forexperimental timeline, see FIG. 7, experiment 1.

Murine Model of Allergic Pulmonary Inflammation: Prevention Studies Micewere treated subcutaneously on day −21 with either M. vaccae (0.1 mg in100 μl of sterile saline), M. vaccae lipid preparation (1, 2, or 5 μg in100 μl of buffer) or with sterile buffer alone. On days 0 and 12, micewere sensitized by intraperitoneal (i.p.) injection of 10 μg chicken eggovalbumin (Grade V, Sigma-Aldrich, UK) in 100 μl of alum gel (AMS,Abingdon, UK). On days 19 and 21, mice were challenged intratracheally(i.t.) with 50 μl of 10 μg/ml ovalbumin in sterile saline solution (FIG.7, Experiments 2-4).

Murine Model of Allergic Pulmonary Inflammation: Treatment Studies

In separate studies, 3 weeks prior to treatment with M. vaccae (0.1 mgin 100 μl of sterile saline), M. vaccae lipid preparation (5 μg in 100μl of buffer) or with sterile buffer alone, animals received two earlierovalbumin and alum injections 12 days apart (on days −42 and −30) todetermine the therapeutic potential of treatment with M. vaccae and itslipid preparations (FIG. 7, Experiments 5 and 6). In some experimentsthe effects of different doses of M. vaccae lipid preparation (5 μg, 2μg, 1 μg and 0.1 μg) were investigated. Mice were sacrificed 24 hoursafter the second i.t. antigen challenge by i.p. injection of sodiumpentobarbital (240 mg/kg, Animal Care, York, UK). The trachea wascannulated and the BAL fluid was collected by washing three times with0.3 ml of RPMI supplemented with 50 U/ml penicillin and 50 μg/mlstreptomycin (Invitrogen). The number of cells recovered was determinedusing a Neubauer chamber. Differential cell counts for each BAL samplewere obtained from slide cytospin (Cytospin 3, Shandon Scientific,Cheshire, UK) stained with Wright-Giemsa (Sigma-Aldrich). A differentialcount of 200 cells was performed using standard histological criteria.The remaining BAL fluid was centrifuged and the supernatant stored at−20° C. for cytokine analysis. Concentrations of IL-10 and IL-5 weremeasured using commercially available ELISA kits.

In Vitro splenocyte Culture

Spleens from mice from each treatment group were pooled and a singlecell suspension was prepared. Erythrocytes were removed by hypotoniclysis. Cells were washed and resuspended in culture media containingRPMI, 10% fetal calf serum, 2 mM glutamine, 50 U/ml penicillin and 50μg/ml streptomycin. Splenocytes (10×10⁶/ml) were stimulated in vitrowith phosphate-buffered saline (PBS; Invitrogen), ovalbumin (50 μg/ml)for antigen-specific stimulation, and with plate-bound anti-CD3 (0.5μg/ml, Pharmingen, Oxford, UK) for polyclonal activation of T cells.Supernatants were collected 72 hours later for analysis of cytokineconcentrations. Concentrations of 1L-10, IL-5 and IFN-γ were measuredusing commercially available ELISA kits.

Synthesis of 10(Z)-hexadecenoic acid; (10Z)-hexadec-10-enoic acid (CASNo. 2511-97-9)

Unless otherwise noted, reagents were obtained commercially and usedwithout further purification. Dichloromethane (CH₂Cl₂) was distilledover calcium hydride (CaH₂) under a nitrogen atmosphere. Tetrahydrofuran(THF; (CH₂)₄O) was distilled from sodium-benzophenone under a nitrogenatmosphere. Thin-layer chromatography analysis of reaction mixtures wasperformed on Dynamic Adsorbents, Inc., silica gel F-254 TLC plates.Flash chromatography was carried out on Zeoprep 60 ECO silica gel. ¹Hspectra were recorded with a Varian INOVA 500 spectrometer. Compoundswere detected by monitoring UV absorbance at 254 nm.

To a 5 mL sealed tube containing 1-heptene (0.50 mL, 3.55 mmol), methyl10-undecenoate (0.080 mL, 0.36 mmol) and 0.35 mL THF was added to aGrubbs Z-selective metathesis catalyst (2.2 mg, 3.48 μmol,Sigma-Aldrich, Cat. No. 771082). The reaction was stirred at 45° C. for8 h before cooling to room temperature. The slurry was filtrated througha short plug of silica gel and concentrated. The obtained oil wasdissolved in 1.0 mL THF. The solution was cooled to 0° C., then9-borabicyclo[3.3.1]nonane (9-BBN) solution in THF (1.28 mL, 0.50 M,0.64 mmol) was added. After 2 h stirring at 0° C., the reaction wasquenched with 60 μL EtOH, then 1.5 mL pH 7 potassium phosphate bufferand 1.5 mL 30% H₂O₂. The mixture was stirred at room temperature for 12h, then extracted with 5 mL EtOAc three times. The combined organiclayers were washed with 4 mL saturated Na₂S₂O₃ and 3 mL brine, thendried over Na₂SO₄, filtered and concentrated. To the crude oil in 1.0 mLTHF was added LiOH monohydrate (38 mg, 0.90 mmol) in 1.0 mL water. After2 h, the reaction solution was cooled to 0° C. before addition of 0.91mL 1.0 N HCl. After being concentrated under reduced pressure, theaqueous solution was saturated with NaCl and extracted with 3 mLdichloromethane three times. The combined organic layers were dried overNa₂SO₄, filtered and concentrated. Purification by flash chromatography(2:1:1 hexanes/dichloromethane/diethyl ether) provided(10Z)-hexadec-10-enoic acid (0.022 g, 90%) as a colourless oil. ¹H NMR(500 MHz, CDCl₃): δ5.48-5.22 (m, 2H), 2.35 (t, J=7.5 Hz, 2H), 2.01 (q,J=6.6 Hz, 4H), 1.63 (p, J=7.4 Hz, 2H), 1.35-1.15 (m, 16H), 0.88 (t,J=6.9 Hz, 3H).

Murine Peritoneal Macrophage Isolation and Screening

Murine peritoneal macrophages were isolated as previously described(Zhang, X. et al. (2008)). Briefly, mice received one 1 ml i.p.injection of 3% thioglycollate medium (Cat. No. 9000-294, VWR, Radnor,Pa., USA). Ninety-six hours later, macrophages were collected in DPBS(Cat. No. 14190136, Invitrogen, Carlsbad, Calif., USA). Cells werecentrifuged and resuspended in DMEM/F-12 (Cat. No. 10565018, Invitrogen)supplemented to be 10% (v/v) fetal bovine serum (Cat. No. 16000036,Invitrogen) and 1% penicillin/streptomycin (Cat. No. 15140148,Invitrogen). One mouse yielded enough cells for one experimentalreplicate. 1×10⁵ cells/well were allowed to adhere for 1.5 h beforebeing washed with DPBS. 10(Z)-hexadecenoic acid was dissolved inDMEM/F-12 with 0.5% (v/v) dimethyl sulfoxide (Cat. No. D8418,Sigma-Aldrich). The macrophages were incubated with either10(Z)-hexadecenoic acid (0.4 μM, 4 μM, 20 μM, 100 μM, 500 μM, 1000 μM)or DMEM/F-12 for 1h before being stimulated with 1 μg/mllipopolysaccharide (serotype 0127:B8, Sigma-Aldrich, St. Louis, Mo.,USA). Culture supernatants were collected at 6, 12, and 24 hpost-stimulation.

Cytokine Measurements

Cell culture supernatants from freshly isolated peritoneal macrophageswere diluted 1:200, and IL-6 was measured using sandwiched ELISA (Cat.No. 431304, Biolegend, San Diego, Calif., USA). All samples weremeasured in duplicate.

Cytotoxicity Assay

Cytotoxicity was determined using the sulforhodamine B (SRB)colorimetric assay, as previously described (Vichai and Kirtikara,2006). Briefly, without removing the culture media, cells were fixed byadding cold trichloroacetic acid and incubated at 4° C. for 1 h. Theplates were washed with slow-running tap water and set out to dryovernight. Then, 0.057% SRB (Cat. No. AC333130050, Fisher, Pittsburgh,Pa., USA), solubilized in 10 mMTris (Cat. No. BP153, Fisher), was addedto each well. After 30 min, plates were washed with 1% acetic acid andset out to dry overnight. SRB was measured at 490 nm on a Synergy HTmicroplate reader (Part Number 7091000, Biotek, Winooski, Vt., USA).Cell viability was expressed as the ratio of experimental and controlgrowth.

Ligands

Rosiglitazone, troglitazone, and WY14643 were obtained from AlexisBiochemicals (San Diego, Calif., USA); ATRA and AM580 fromSigma-Aldrich. In addition, GW9662 was a gift from T. M. Wilson(GlaxoSmithKline, Brentford, United Kingdom).

Statistical Analysis

Results are represented as means±SEM. Data were subjected to a normalitytest and one-way ANOVA or Student's t-tests were performed asappropriate. A two-tailed p value≤0.05 was considered significant.

What is claimed is:
 1. A pharmaceutical composition comprising a fattyacid of at least C11 comprising a double bond between C10 and C11 and atleast one of an adjuvant, a preservative, and a stabiliser.
 2. Thepharmaceutical composition of claim 1, wherein the fatty acid is fromC11 to C24.
 3. The pharmaceutical composition of claim 1, wherein thefatty acid comprises only one double bond.
 4. The pharmaceuticalcomposition of claim 1, wherein the fatty acid is C16
 5. Thepharmaceutical composition of claim 1, wherein the fatty acid is10(Z)-hexadecenoic acid.
 6. The pharmaceutical composition of claim 1,wherein the fatty acid is an ester of 1,2,3-propanetriol with one ormore fatty acids of at least C11, wherein at least one fatty acid has adouble bond at the C10-C11 position.
 7. The pharmaceutical compositionof claim 1, formulated as an injectable liquid.
 8. The pharmaceuticalcomposition of claim 1, formulated in a sterile freeze-dried form thatis reconstituted prior to use.
 9. The pharmaceutical composition ofclaim 1, formulated as sterile slow-release pellets.
 10. A method oftreating sepsis and/or preventing or treating post-sepsis syndromecomprising administration of an effective amount of a fatty acid of atleast C11 comprising a double bond between C10 and C11 to a patient inneed of such treatment.
 11. The method of claim 10, wherein the fattyacid is from C11 to C24.
 12. The method of claim 10, wherein the fattyacid comprises only one double bond.
 13. The method of claim 10, whereinthe fatty acid is C16
 14. The method of claim 13, wherein the fatty acidis 10(Z)-hexadecenoic acid.
 15. The method of claim 10, wherein thefatty acid is an ester of 1,2,3-propanetriol with one or more fattyacids of at least C11, wherein at least one fatty acid has a double bondat the C10-C11 position.
 16. The method of claim10, further comprisingthe separate, sequential, or simultaneous administration of anantibacterial, anti-fungal, or anti-viral molecule known to eradicatethe infective agent causing sepsis.
 17. The method of claim 10, whereinthe fatty acid molecule is administered via the parenteral route or theenteral route.
 18. The method of claim 17, wherein the parenteral routeis selected from subcutaneous, intradermal, subdermal, intraperitoneal,intravenous, and intravesicular injection.
 19. The method of claim 8,wherein the fatty acid molecule is administered via intravenousinjection.
 20. The method of claim 10, wherein the fatty acid moleculeis administered via the intratracheal route.